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Updated: Jul 18, 2026
Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro
Published on: February 15, 2022
Proteomic analysis of vascular smooth muscle cells treated with ouabain
1Department of Medical Genetics, St. Justine Hospital, Montreal, Quebec, Canada.
Abstract:
Apoptosis of vascular smooth muscle cells (VSMC) plays an important role in remodeling the vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Apoptosis in VSMC can be inhibited by inversion of the intracellular [Na+]/[K+] ratio after the sustained blockage of the Na+,K+-ATPase by ouabain. Using two-dimensional gel electrophoresis followed by tandem mass spectroscopy, we compared proteomes of control VSMC and of those with ouabain-inhibited Na+,K+-ATPase and found that ouabain treatment led to overexpression of numerous soluble and membrane-bound proteins. Among proteins, which showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 superfamily and a marker for cellular mortal and immortal phenotypes. Further experiments showed that mortalin RNA and protein levels are induced in ouabain-treated VSMC, and that transient transfection of cells with mortalin cDNA inhibited serum deprivation-induced apoptosis via inactivation of the tumor suppressor gene, p53.
Insights
Vascular smooth muscle cell (VSMC) apoptosis contributes to high blood pressure. Ouabain treatment inhibits VSMC apoptosis by increasing mortalin, a heat shock protein, which inactivates the tumor suppressor p53.
Area of Science:
- Cardiovascular Biology
- Cellular Biology
- Molecular Medicine
Background:
- Vascular smooth muscle cell (VSMC) apoptosis is crucial in blood vessel wall remodeling, impacting blood pressure and cardiovascular health.
- Sustained Na+,K+-ATPase blockage by ouabain can inhibit VSMC apoptosis by altering intracellular ion ratios.
- Understanding the molecular mechanisms behind ouabain's effect on VSMC apoptosis is vital for cardiovascular disease research.
Purpose of the Study:
- To investigate the proteomic changes in VSMC following ouabain-induced Na+,K+-ATPase inhibition.
- To identify key proteins involved in the anti-apoptotic effects of ouabain in VSMC.
- To elucidate the role of mortalin in mediating the inhibition of apoptosis in VSMC.
Main Methods:
- Proteomic analysis using two-dimensional gel electrophoresis and tandem mass spectrometry to compare control and ouabain-treated VSMC.
- Quantification of mortalin RNA and protein levels in response to ouabain.
- Functional studies involving transient transfection with mortalin cDNA to assess its effect on apoptosis.
Main Results:
- Ouabain treatment resulted in the overexpression of numerous proteins in VSMC.
- Mortalin (GRP75/PBP-74), a heat shock protein, was identified as a highly expressed protein after ouabain treatment.
- Increased mortalin levels inhibited serum deprivation-induced apoptosis in VSMC by inactivating the p53 tumor suppressor gene.
Conclusions:
- Mortalin plays a significant role in inhibiting VSMC apoptosis induced by ouabain.
- The mortalin-p53 pathway is a key mechanism by which ouabain exerts its anti-apoptotic effects in VSMC.
- Targeting mortalin may offer a novel therapeutic strategy for managing cardiovascular conditions associated with VSMC apoptosis.
